Characterizing the glycoforms of RNase 1, and, Developing fluorogenic indicators of
cytosolic entry
Robert G. Presler, Jr.
Prof. Ronald T. Raines
Dept. of Biochemsitry, Integrated Program in Biochemistry (IPiB)
The secreted, RNA-degrading protein bovine ribonuclease A (RNase A) is perhaps the most studied
enzyme of the 20th century. RNase A was the first enzyme to have its chemical mechanism
understood, it was among the first protein structures to be solved, and its stability is legendary
(classically, RNase A purification relies upon its ability to survive brutal acidification and heatdenaturation). Nevertheless, despite half a century of scientific intimacy with this 14-kDa bovine
protein, much of the biology and biochemistry of the native human homolog (RNase 1) remains
unclear.
Rigorous characterization of native human RNase 1 has proven to be challenging. The current
inability to efficiently separate and study individual glycoforms of native RNase 1 greatly limits
scientific inquiry. To illustrate this point, bovine RNase A can only be decorated with a single
glycan (to yield “RNase B”); human RNase 1, however, can potentiate as many as seven individual
glycoforms. Thus, whereas RNases A and B can be easily separated, RNase 1 can only be subdivided
into four fractions: (I) mono-, (II) di-, (III) tri-, and (IV) unglycosylated. Illustrated aside, fractions
(I) and (II) are actually a mixture of individual glycoforms. Herein, a strategy is described to
produce all seven RNase 1 glycoforms, thus enabling rigorous biochemical and cell biological
analyses.
Cell biology has been revolutionized by the ability to detect otherwise invisible macromolecules by
using small-molecule fluorophores. For example, esterase-labile latent fluorophores have enabled
the kinetic resolution of RNase A endosomal entry. A key step in RNase A internalization, cytosolic
entry, has yet to be sufficiently resolved. In this research effort, a strategy is described for the
generation of cytosol-sensing latent fluorophores.